Worm Breeder's Gazette 13(3): 96 (June 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Molecular Analysis of lin-19 (a member of a novel gene family) & lin-23 (encoding a protein with ß-transducin repeats and a Xenopus ortholog)

Edward T. Kipreos, Edward M. Hedgecock

Department of Biology, Johns Hopkins University, Baltimore, MD 21218

In the absence of lin-19 and lin-23 gene function, extra cells are generated in multiple tissues, suggesting that the wild-type gene products function to limit cell proliferation (WM 93, p.244). Both lin-19 and lin-23 have been cloned by transformation rescue of the mutant worms.

lin-19 encodes an 822 amino acid protein ( LIN-19 ).The protein has a potential nuclear translocation signal, but no other discernible protein motifs. By searching the EST database, LIN-19 was found to belong to a family of uncharacterized proteins. The family members are 20 to 40% identical to each other. There are at least three human, one rice, and two C. elegans members ( lin-19 and lnh-1 ,lin-nineteen homolog-1). Interestingly, LIN-19 is most closely related to the rice homolog, while LNH-1 is most closely related to a human homolog, suggesting that this gene family was created before the divergence of plants and animals. The two lin-19 mutant alleles are nonsense mutations. For one allele, the mutation causes loss of 62% of the Lin-19 coding domain, and is probably a null allele.

in situ RNA analysis of embryos (Geraldine Seydoux, personal communication) shows that lin-19 has a pulse of zygotic expression from the 12-15 cell stage to the 60 cell stag. All cells stain with the exception of the germ lineage cells, P3 and P4 .No RNA is detected before or after this stage in the embryo. We are currently engaged in determining the full expression pattern of the RNA and protein through development.

lin-23 encodes a 655 amino acid protein with a putative nuclear translocation signal. The central domain of LIN-23 comprises seven WD40 (ß-transducin) repeats. First described in the transducin G-ß subunit, this ~40 amino acid motif has since been found, typically as 5-8 tandem copies, in functionally diverse nuclear and cytoplasmic proteins. Specific roles of WD40 repeats are unknown in most cases, but it is proposed that they mediate protein-protein interactions. All four known lin-23 alleles are nonsense mutations located in conserved tryptophans of the WD40 repeats. This clustering of the nonsense mutations, allowing the translation of 65 to 77% of the LIN-23 protein, suggests that these are not null alleles.

An apparent ortholog of LIN-23 is ß-TrCP, a Xenopus protein. Both proteins have seven WD40 repeats that are 82% identical, and the proteins are 33% identical in the N-terminal domain In contrast, the next most similar protein, CDC4 of S. cerevisiae, is only 31% identical to LIN-19 in the WD40 domain and is unrelated in the N-terminal domain.

ß-TrCP was discovered by its ability to functionally complement the loss of CDC15 activity in the yeast S. cerevisiae (Spevak et al., 1993, M.C.B., vol.13, p. 49-53). CDC15 is a protein kinase; cdc15 -mutants arrest at late anaphase of mitosis, with condensed chromosomes located at the separated spindle pole bodies. The function of ß-TrCP in Xenopus is presently unknown.